Sheila Aparecida Coelho Siqueira

The impact of clinical and genetic screenings on the management of the multiple endocrine neoplasia type 1

PURPOSE To perform clinical and genetic screening for multiple endocrine neoplasia type 1 (MEN1) in patients at the Academic Hospital of the University of São Paulo School of Medicine, and to analyze its impact on clinical management of patients with MEN1. METHODS The clinical diagnosis of MEN1 was made in accordance with the Consensus on multiple endocrine neoplasias. Mutation analysis of the entire MEN1 tumor suppressor gene and genetic screening of at-risk family members were performed by direct sequencing. To analyze the implementation of genetic diagnosis, the studied patients were separated into 3 groups: MEN1 index cases (group I), clinically diagnosed MEN1 cases (group II), and genetically diagnosed MEN1 cases (group III). RESULTS In total, 154 individuals were clinically and genetically studied. We identified 12 different MEN1 mutations. Fifty-two MEN1 cases were identified: 13 in group I, 28 in group II, and 11 in group III. The mean age in group III (27.0 years) was significantly lower than in groups I (39.5 years) and II (42.4 years; P = 0.03 and P = 0.01, respectively). Patients in groups I and II mostly presented 2 or 3 MEN1-related tumors, while 81.8% of those in group III presented 1 or no MEN1-related tumor. Additionally, in group III, 45.4% of cases were asymptomatic, and no metastasis or death was verified. Surveillance for MEN1 mutations allowed the exclusion of 102 noncarriers, including a case of MEN1 phenocopy. CONCLUSION Our data supports the benefits of clinical and genetic screening for multiple endocrine neoplasia type 1 in the management of this syndrome.

Cigarette smoking and high‐risk HPV DNA as predisposing factors for high‐grade cervical intraepithelial neoplasia (CIN) in young Brazilian women

BACKGROUND This cross-sectional study was designed to evaluate the role of cigarette smoking and high-risk HPV types as risk factors of CIN 2 and 3 in young, sexually active Brazilian women. MATERIALS AND METHOD A series of 100 consecutive women with abnormal Pap smears were recruited, subjected to colposcopy, punch biopsy, and questionnaire for their social, sexual and reproductive factors. Of these, 77 women between 20 and 35 years of age (median 26.5 years) with biopsy-confirmed CIN 1 or CIN 2 and 3, were enrolled in this study. Representative samples from the exocervix and endocervix were obtained for HPV testing with the Hybrid Capture HPV-DNA assay, including the probes for the oncogenic HPV types (16, 18, 31, 33, 35, 45, 51, 52 and 56). RESULTS The overall rate of CIN 2 and 3 was 23/77 (29.8%). The women with CIN 1, 2 and 3 did not differ from each other with regard to their age, race, schooling, marital status, lifetime number of sexual partners, age at first intercourse, use of oral contraceptives, or parity. However, current cigarette smoking was strongly associated with CIN 2 and 3 (p<0.001), and among smokers, the risk of high-grade CIN increased in parallel with the time of exposure (years of smoking) (p=0.07). HPV-DNA of the oncogenic types was detected in 43 (56%) women, the risk of being HPV DNA-positive was significantly higher in CIN 2 and 3 as compared with CIN 1 (p=0.037). Importantly, the prevalence of high-risk HPV types was significantly higher in cigarette smokers than in non-smokers (p=0.046). CONCLUSIONS The results indicate that the severity of CIN lesions was clearly related to two fundamental risk factors: 1) high-risk HPV types, and 2) current cigarette smoking. These two risk factors were closely interrelated in that the high-risk HPV types were significantly more frequent in current smokers than in non-smokers, suggesting the possibility of a synergistic action between these two risk factors in cervical carcinogenesis.

Multiple endocrine neoplasia type 1 in Brazil: MEN1 founding mutation, clinical features, and bone mineral density profile

OBJECTIVE Only few large families with multiple endocrine neoplasia type 1 (MEN1) have been documented. Here, we aimed to investigate the clinical features of a seven-generation Brazilian pedigree, which included 715 at-risk family members. DESIGN Genealogical and geographic analysis was used to identify the MEN1 pedigree. Clinical and genetic approach was applied to characterize the phenotypic and genotypic features of the family members. RESULTS Our genetic data indicated that a founding mutation in the MEN1 gene has occurred in this extended Brazilian family. Fifty family members were diagnosed with MEN1. Very high frequencies of functioning and non-functioning MEN1-related tumors were documented and the prevalence of prolactinoma (29.6%) was similar to that previously described in prolactinoma-variant Burin (32%). In addition, bone mineral density analysis revealed severe osteoporosis (T, -2.87+/-0.32) of compact bone (distal radius) in hyperparathyroidism (HPT)/MEN1 patients, while marked bone mineral loss in the lumbar spine (T, -1.95+/-0.39), with most cancellous bone, and femoral neck (mixed composition; T, -1.48+/-0.27) were also present. CONCLUSIONS In this study, we described clinically and genetically the fifth largest MEN1 family in the literature. Our data confirm previous findings suggesting that prevalence of MEN1-related tumors in large families may differ from reports combining cumulative data of small families. Furthermore, we were able to evaluate the bone status in HPT/MEN1 cases, a subject that has been incompletely approached in the literature. We discussed the bone loss pattern found in our MEN1 patients comparing with that of patients with sporadic primary HPT.

High Penetrance of Pheochromocytoma Associated with the Novel C634Y/Y791F Double Germline Mutation in the RET Protooncogene

CONTEXT Previous studies have shown that double RET mutations may be associated with unusual multiple endocrine neoplasia type 2 (MEN 2) phenotypes. OBJECTIVE Our objective was to report the clinical features of patients harboring a previously unreported double mutation of the RET gene and to characterize this mutation in vitro. PATIENTS Sixteen patients from four unrelated families and harboring the C634Y/Y791F double RET germline mutation were included in the study. RESULTS Large pheochromocytomas measuring 6.0-14 cm and weighing up to 640 g were identified in the four index cases. Three of the four tumors were bilateral. High penetrance of pheochromocytoma was also seen in the C634Y/Y791F-mutation-positive relatives (seven of nine, 77.7%). Of these, two cases had bilateral tumors, one presented with multifocal tumors, two cases had large tumors (>5 cm), and one case, which was diagnosed with a large (5.5 x 4.5 x 4.0 cm) pheochromocytoma, reported early onset of symptoms of the disease (14 yr old). The overall penetrance of pheochromocytoma was 84.6% (11 of 13). Development of medullary thyroid carcinoma in our patients seemed similar to that observed in patients with codon 634 mutations. Haplotype analysis demonstrated that the mutation did not arise from a common ancestor. In vitro studies showed the double C634Y/Y791F RET receptor was significantly more phosphorylated than either activated wild-type receptor or single C634Y and Y791F RET mutants. CONCLUSIONS Our data suggest that the natural history of the novel C634Y/Y791F double mutation carries a codon 634-like pattern of medullary thyroid carcinoma development, is associated with increased susceptibility to unusually large bilateral pheochromocytomas, and is likely more biologically active than each individual mutation.

ERCC1 protein, mRNA expression and T19007C polymorphism as prognostic markers in head and neck squamous cell carcinoma patients treated with surgery and adjuvant cisplatin-based chemoradiation

Adjuvant cisplatin-based chemoradiation improves survival in HNSCC patients presenting with risk features. ERCC1 (excision repair cross-complementation group 1) is associated with resistance to chemo- and radiation therapy and may have a prognostic value in HNSCC patients. Here we studied ERCC1 expression and the polymorphism T19007C as prognostic markers in these patients. This is a retrospective and translational analysis, where ERCC1 protein expression was evaluated by immunohistochemistry, using an H-score, and mRNA expression was determined by RT-PCR. T19007C genotypes were detected by PCR-RFLP carried out using DNA template extracted from normal lymph nodes. A high H-score was seen in 32 patients (54%), who presented better 5-year overall survival (5-y OS: 50% vs. 18%, HR 0.43, p=0.026). Fifteen out of 45 patients (33%), with high mRNA expression, presented better 5-year overall survival (OS) (86% vs. 30%, HR 0.26, p=0.052). No OS difference was detected among T19007C genotypes. High H-score and mRNA expression remained significant as favorable prognostic factors in a multivariate analysis. Collectively, our results suggest that high ERCC1 expression seems to be associated with better OS rates in HNSCC patients submitted to adjuvant cisplatin-based chemoradiation.

Penetrance of functioning and nonfunctioning pancreatic neuroendocrine tumors in multiple endocrine neoplasia type 1 in the second decade of life.

CONTEXT Data are scarce on the penetrance of multiple endocrine neoplasia type 1 (MEN1)-related nonfunctioning pancreatic neuroendocrine tumors (NF-PETs) and insulinomas in young MEN1 patients. A potential positive correlation between tumor size and malignancy (2-3 cm, 18%; >3 cm, 43%) has greatly influenced the management of MEN1 adults with NF-PETs. OBJECTIVE The aim of the study was to estimate the penetrance of NF-PETs, insulinomas, and gastrinomas in young MEN1 carriers. DESIGN The data were obtained from a screening program (1996-2012) involving 113 MEN1 patients in a tertiary academic reference center. PATIENTS Nineteen MEN1 patients (aged 12-20 y; 16 patients aged 15-20 y and 3 patients aged 12-14 y) were screened for NF-PETs, insulinomas, and gastrinomas. METHODS Magnetic resonance imaging/computed tomography and endoscopic ultrasound (EUS) were performed on 10 MEN1 carriers, magnetic resonance imaging/computed tomography was performed on five patients, and four other patients underwent an EUS. RESULTS The overall penetrance of PETs during the second decade of life was 42% (8 of 19). All eight PET patients had NF-PETs, and half of those tumors were multicentric. One-fifth of the screened patients (21%; 4 of 19) harbored at least one large tumor (>2.0 cm). Insulinoma was detected in two NF-PET patients (11%) at the initial screening; gastrinoma was not present in any cases. Six of the 11 (54%) screened patients aged 15-20 years who underwent an EUS had NF-PETs. Potential false-positive EUS results were excluded based on EUS-guided biopsy results, the reproducibility of the NF-PET findings, or the observation of increased tumor size during follow-up. Distal pancreatectomy and the nodule enucleation of pancreatic head tumors were conducted on three patients with large tumors (>2.0 cm; T2N0M0) that were classified as grade 1 neuroendocrine tumors (Ki-67<2%). CONCLUSIONS Our data demonstrated high penetrance of NF-PETs in 15- to 20-year-old MEN1 patients. The high percentage of the patients presenting consensus criteria for surgery for NF-PET alone or NF-PET/insulinoma suggests a potential benefit for the periodic surveillance of these tumors in this age group.

Existence of a potential neurogenic system in the adult human brain

BackgroundPrevailingly, adult mammalian neurogenesis is thought to occur in discrete, separate locations known as neurogenic niches that are best characterized in the subgranular zone (SGZ) of the dentate gyrus and in the subventricular zone (SVZ). The existence of adult human neurogenic niches is controversial.MethodsThe existence of neurogenic niches was investigated with neurogenesis marker immunostaining in histologically normal human brains obtained from autopsies. Twenty-eight adult temporal lobes, specimens from limbic structures and the hypothalamus of one newborn and one adult were examined.ResultsThe neural stem cell marker nestin stained circumventricular organ cells and the immature neuronal marker doublecortin (DCX) stained hypothalamic and limbic structures adjacent to circumventricular organs; both markers stained a continuous structure running from the hypothalamus to the hippocampus. The cell proliferation marker Ki-67 was detected predominately in structures that form the septo-hypothalamic continuum. Nestin-expressing cells were located in the fimbria-fornix at the insertion of the choroid plexus; ependymal cells in this structure expressed the putative neural stem cell marker CD133. From the choroidal fissure in the temporal lobe, a nestin-positive cell layer spread throughout the SVZ and subpial zone. In the subpial zone, a branch of this layer reached the hippocampal sulcus and ended in the SGZ (principally in the newborn) and in the subiculum (principally in the adults). Another branch of the nestin-positive cell layer in the subpial zone returned to the optic chiasm. DCX staining was detected in the periventricular and middle hypothalamus and more densely from the mammillary body to the subiculum through the fimbria-fornix, thus running through the principal neuronal pathway from the hippocampus to the hypothalamus. The column of the fornix forms part of this pathway and appears to coincide with the zone previously identified as the human rostral migratory stream. Partial co-labeling with DCX and the neuronal marker βIII-tubulin was also observed.ConclusionsCollectively, these findings suggest the existence of an adult human neurogenic system that rises from the circumventricular organs and follows, at minimum, the circuitry of the hypothalamus and limbic system.

Cytologic findings of epithelioid angiosarcoma of the thyroid: A case report

BACKGROUND Angiosarcoma of the thyroid is a rare and aggressive tumor and occurs mainly in patients from central Europe, especially the alpine region. The fine needle aspiration findings of a keratin-positive epithelioid angiosarcoma of the thyroid occurring in a nonmountainous area in South America is described. CASE A 65-year-old male from São Paulo, Brazil, presented with a mass in the anterior part of the neck with progressive enlargement for three months. The cytologic findings on the fine needle aspirate were a cellular smear composed of single cells and small clusters of neoplastic cells, oval and round. Cell borders were indistinct, and the cytoplasm was vacuolated. The nuclei were eccentrically located, with irregular nuclear membranes; single, prominent nucleoli; and a coarse chromatin pattern. Features suggestive of intracytoplasmic lumens were identified. Open surgical biopsy demonstrated a tumor infiltrating the thyroid gland and composed of large, round, atypical epithelioid cells lining vascular spaces. These neoplastic cells were immunoreactive for AE1:AE3, CK7, vimentin, CD31 and factor VIII. CONCLUSION Epithelioid angiosarcoma should be considered in the differential diagnosis of epithelioid neoplasms of the thyroid. An immunohistochemical panel should include vascular markers even in the presence of immunoreactivity for epithelial markers.

Penetrance and clinical features of pheochromocytoma in a six-generation family carrying a germline TMEM127 mutation.

CONTEXT The phenotype of familial pheochromocytoma (PHEO) associated with germline TMEM127 mutations (TMEM127-related PHEO) has not been clearly defined. OBJECTIVE This study aimed to investigate the penetrance, full phenotypic spectrum and effectiveness of clinical/genetic screening in TMEM127-related PHEO. DESIGN, SETTING, AND PARTICIPANTS Clinical and genetic screening, and genetic counseling were offered to 151 individuals from a six-generation family carrying a TMEM127 germline mutation in a referral center. INTERVENTION AND MAIN OUTCOME MEASURES TMEM127 genetic testing was offered to at-risk relatives and clinical surveillance for pheochromocytoma was performed in mutation-positive carriers. RESULTS Forty seven individuals carried the c.410-2A>C TMEM127 mutation. Clinical data were obtained from 34 TMEM127-mutation carriers followed up for 8.7 ± 8.1 years (range, 1-20 y). Pheochromocytoma was diagnosed in 11 carriers (32%) at a median age of 43 years. In nine patients, symptoms started at 29 years (range, 10-55 y) and two cases were asymptomatic. Tumors were multicentric in five (45%) and bilateral in five (45%) patients. Six patients (54%) had at least one adrenomedullary nodule less than 10 mm. No paragangliomas, distant metastases, or other manifestations were detected. Cumulative penetrance of pheochromocytoma was 0% at 0-20 years, 3% at 21-30 years, 15% at 31-40 years, 24% at 41-50 years, and 32% at 51-65 years. The youngest case was diagnosed at 22 years and the earliest symptoms were reported at age 10. CONCLUSIONS Tumor multicentricity, nodular adrenomedullary hyperplasia, and the occurrence of symptoms more than a decade earlier than the age at diagnosis are novel findings in TMEM127-related PHEO. The high penetrance of pheochromocytoma in this condition validates the benefits of genetic testing of at-risk relatives. We thus recommend that TMEM127 genetic testing should be offered to at-risk individuals at age 22 years and mutation carriers should undergo clinical surveillance annually.

Serpin Peptidase Inhibitor Clade A Member 1 as a Potential Marker for Malignancy in Insulinomas

Purpose: The biological behavior of insulinomas cannot be predicted based on histopathologic criteria in which the diagnosis of malignancy is confirmed by the presence of metastases. In this study, microarray and quantitative real-time reverse transcription-PCR were applied to identify differentially expressed genes between malignant and nonmalignant insulinomas to search for useful biomarkers to recognize the metastatic potential of insulinomas. Experimental Design: CodeLink human bioarrays were used to analyze differences in ∼20,000 genes between six well-differentiated endocrine tumors of benign behavior compared with one well-differentiated endocrine carcinoma (WDEC) and three metastases of endocrine carcinomas (MEC). Quantitative real-time reverse transcription-PCR was used to validate differential expressions of five genes in a series of 35 sporadic insulinomas. Serpin peptidase inhibitor clade A member 1 (SERPINA1; α-1-antitrypsin) expression, identified as up-regulated in malignant insulinomas, was also evaluated by immunohistochemistry. Results: Analysis of microarray data resulted in 230 differentially expressed genes. Gene Ontology analysis identified serine-type endopeptidase activity and serine-type endopeptidase inhibitor activity as pathways presenting significant differential expression. Protease serine 2 and complement factor B (from serine-type endopeptidase activity pathway) were respectively confirmed as up-regulated in well-differentiated endocrine tumors of benign behavior (WDET) and in WDEC/MEC. Angiotensinogen and SERPINA1 (from serine-type endopeptidase inhibitor activity pathway) were confirmed as up-regulated in WDEC/MEC. SERPINA1 was shown to be expressed in 85.7% of malignant versus 14.3% of nonmalignant insulinomas by immunohistochemistry. Conclusions: Our data are consistent to the possibility that SERPINA1 is a marker of malignancy in insulinomas. Given the widespread availability of antibody anti-α-1-antitrypsin in pathology services, SERPINA1 expression evaluation might be of clinical utility in recognizing patients more likely to develop an aggressive presentation.